Organoplatinum Compounds as Anion‐Tuneable Uphill Hydroxide Transporters

Abstract Active transport of ions uphill, creating a concentration gradient across a cell membrane, is essential for life. It remains a significant challenge to develop synthetic systems that allow active uphill transport. Here, a transport process fuelled by organometallic compounds is reported that creates a pH gradient. The hydrolysis reaction of PtII complexes results in the formation of aqua complexes that established rapid transmembrane movement (“flip‐flop”) of neutral Pt−OH species, leading to protonation of the OH group in the inner leaflet, generating OH− ions, and so increasing the pH in the intravesicular solution. The organoplatinum complex effectively transports bound hydroxide ions across the membrane in a neutral complex. The initial net flow of the PtII complex into the vesicles generates a positive electric potential that can further drive uphill transport because the electric potential is opposed to the chemical potential of OH−. The OH− ions equilibrate with this transmembrane electric potential but cannot remove it due to the relatively low permeability of the charged species. As a result, effective hydroxide transport against its concentration gradient can be achieved, and multiple additions can continuously drive the generation of OH− against its concentration gradient up to ΔpH>2. Moreover, the external addition of different anions can control the generation of OH− depending on their anion binding affinity. When anions displayed very high binding affinities towards PtII compounds, such as halides, the external anions could dissipate the pH gradient. In contrast, a further pH increase was observed for weak binding anions, such as sulfate, due to the increase of positive electric potential.

Compound 4,4′-Bis(trans-Pt(PEt3)2OTf)benzophenone 1. The platinum compound 4,4′-bis(trans-Pt(PEt3)2-OTf)benzophenone (1) was synthesized from AgOSO2CF3 and the Pt(II) bromide compound (5), which is made by 4,4′-dibromidobenzophenone and Pt(PEt3)4 according to literature procedures. [1] A flask was charged with 4,4'-bis(trans-Pt(PEt3)2Br)benzophenone (5, 120 mg, 0.1 mmol) under nitrogen and dissolved in CH2Cl2 (15 mL). The reaction was then cooled to 0 ℃ with an ice bath, and AgOTf (54.0 mg, 0.21 mmol) was added. The reaction was allowed to stir for 3 h, and the AgBr precipitate was filtered off. The solvent volume was reduced to 4 mL, and the product precipitated with hexanes (15 mL). The compound was isolated as a white powder in an 82% yield. (98.4 mg): Compound 1,4-Bis(trans-Pt(PEt3)2OTf)benzene 2. To a stirred solution of the previously reported 4,4'-bis(trans-Pt(PEt3)2I)benzene [2] (0.211 g, 0.177 mmol) in CH2Cl2 (10 mL) at 0 °C was added, AgOTf (0.049 g, 0.19 mmol) in the dark and under a nitrogen atmosphere; a light-yellow suspension immediately formed. After ∼2 h of stirring, the room-temperature suspension was cannula filtered to remove the AgI, and to the filtrate was added an equal volume of dry diethyl ether. The resulting solution was subsequently placed in the freezer for 1−2 days. The white, crystalline solid that formed was then filtered under nitrogen and dried in vacuo with slight heating (40 °C): yield 0.195 g (89%); 1 H-NMR (CD2Cl2) δ 6.79 (s, 4H, 3  Compound 2,9-bis[trans-Pt(PEt3)2(OTf)]phenanthrene 3. 2,9-bis[trans-Pt(PEt3)2Br]phenanthrene [3] (120 mg, 0.1 mmol) and AgOTf (54.0 mg, 0.21 mmol) were taken in dichloromethane (3 mL) in a 10 mL round bottom flask. The reaction mixture was stirred at room temperature for 24 h under a nitrogen atmosphere in the dark. An off-white solid was formed, which was filtered off through a glass fiber filter, and the solvent was concentrated under reduced pressure. Cold n-pentane was slowly added to this solution to afford white precipitate. This precipitate was washed thoroughly with n-pentane thrice (3 × 10 mL). The supernatant was decanted, and the solid was dried in vacuo to afford a white microcrystalline product (Yield: 87%). Compound 2,9-bis[trans-Pt(PEt3)2(NO3)]phenanthrene 4. 2,9-bis[trans-Pt(PEt3)2Br]phenanthrene [3] (145 mg, 0.12 mmol) and AgNO3 (200 mg, 1.18 mmol) were placed in a 2-dram vial followed by dichloromethane (3 mL). The reaction was stirred in the dark at room temperature for 24 h. A clear solution with a heavy creamy precipitate resulted, the precipitate was filtered off, and the solvent was removed under a flow of nitrogen. The residue was redissolved in a minimal amount of dichloromethane, and then n-pentane was carefully added to precipitate the residual AgNO3, but not the product. The cloudy solution that resulted was filtered through a glass fiber filter, and the product was then precipitated by the addition of more n-pentane. The supernatant was decanted, and the product was dried in vacuo overnight. Yield: 120 mg (86%

S1.3.1 General methodology
HPTS assays were conducted using POPC vesicles (mean diameter 200 nm) loaded with the pH-sensitive fluorescence dye HPTS (1 mM). The HPTS-loaded POPC LUVs were prepared as follows. A chloroform solution of POPC was evaporated in a round-bottom flask, and the lipid film formed was dried under vacuum for at least 6 h. Then, the lipid film was hydrated by vortexing with an internal solution containing HPTS (1 mM) buffered with 10 mM HEPES. The HEPES buffer of different pH was prepared by dissolving an appropriate amount of solid HEPES (10 mM) and salt in deionized water. The lipid suspension was subjected to nine freeze/thaw cycles, where the suspension was alternatingly allowed to freeze in a liquid nitrogen bath, followed by thawing in a water bath. The lipid suspension was allowed to age for 30 min at room temperature and was subsequently extruded 25 times through a Whatman® Nucleopore™ 200 nm polycarbonate membrane using an extruder set (Avanti Polar Lipids, Inc). The unentrapped HPTS was removed by size exclusion chromatography on a Sephadex® G-25 column using an external solution eluent that does not contain HPTS. The lipid solution obtained after Sephadex® was diluted to a standard volume (usually 5 mL) using the buffered HEPES external solution to obtain a lipid stock of known concentration. For each test, the lipid stock was diluted with the external buffer solution to a standard volume (2.5 mL) to afford a solution with a lipid concentration of 0.1 mM in a disposable PMMA cuvette with a stirrer bar. The LUV suspension was allowed to equilibrate at 25 °C for ~2 min in the cuvette cell holder of the spectrofluorometer. The fluorescence intensities of HPTS (λex 403 nm | λem 510 nm) and the deprotonated HPTS (λex 460 nm | λem 510 nm) were recorded simultaneously at 3 s time-intervals. All ionophores and fatty acids were dissolved and added as DMSO solutions (5 µL) to the LUV suspension.

S1.3.2 HPTS calibration
Determination of pH by HPTS is based on the ratio of fluorescence intensity at the emission wavelength of 510 nm measured at two excitation wavelengths: one at 460 nm, indicating the level of the unprotonated ionized HPTS 8-hydroxy group (which is pH-dependent), and one at 403 nm, indicating the level of total HPTS in the system irrespective of whether its 8-hydroxy group is ionized or not; thus, 403 nm is the pH-independent isosbestic point. The 460/403 nm excitation ratio (emission at 510 nm) is a direct measure of the level of HPTS ionization. The pH was calculated from the 460/403 nm excitation ratio of the fluorescence intensities using Eq. (1): where R is the F460/F403 ratio (with F460 representing the fluorescence intensity of nonprotonated charged HPTS and F403 representing the fluorescence intensity of the total HPTS in the experimental system [O − plus OH forms]), pKa is the apparent pKa of the fluorescent probe, and Ra and Rb are the fluorescence intensity ratios of the protonated and unprotonated forms of the probe, respectively.
It is known that the pKa of HPTS is dependent on the ionic strength and medium composition.
[4] Therefore, the apparent pKa of HPTS is likely to be different in a bulk aqueous solution compared to encapsulated inside the LUVs (due to the ionic charged surface of lipid headgroups) and in the presence of detergent micelles. For the calibration of HPTS fluorescence response to pH, fluorescence measurements were conducted using the same experimental conditions as for the transport studies.
(2) Figure S1. The HPTS pH calibration gave good fitting relationships for the ratiometric intensity F460/F403 as a function of pH (Figures S1). In the following experiments, the pH values for the transport studies can be calculated from the ratiometric intensity F460/F403 according to Eq.
(1); where the intravesicular pH (pHin) and the bulk pH after the addition of detergent to lyse the vesicles (pHlysed) can be obtained from the corresponding apparent pKa, Rb, and Ra values according to the standard curve generated beforehand from the treatment with detergent ( Figure. S1 and Eq. (2)).

S1.3.3 HPTS interference in aqueous solution
In order to exclude the possible interference from the interaction between metal compounds with HPTS, a fluorescence titration experiment between compound 1 and HPTS was conducted. Based on the intravesicular volume calculation shown below (in section S2.2.1), the fraction of interior volume to total volume is 0.059% for LUVs with 200 nm diameter, at a lipid concentration of 0.1 mM.
Since the intravesicular concentration of HPTS is 1 mM, therefore the overall concentration of HPTS in each fluorescence measurement is approximately ~0.59 µM.
For the study of HPTS interference with metal compounds in an aqueous medium, fluorescence measurements were conducted on a solution of HPTS (0.5 μM) buffered to pH 7.0 with HEPES (10 mM) with the addition of a DMSO solution of compound 1 (5 μL) with increasing concentration from 0.5 μM to 5 μM. Moreover, fluorescence measurements were also conducted on a solution of HPTS (0.5 μM) with NaCl (40 mM) buffered to pH 7.0 with HEPES (10 mM) with the addition of a DMSO solution of compound 1 (5 μL) with increasing concentration from 0.5 μM to 5 μM to further explore the interference of slat. In this study, the HPTS ratiometric intensity F460/F403 is reported as the relative response to the HPTS ratiometric intensity without the presence of additives calculated using the following Eq. (3): where Ir(rel.) is the relative HPTS ratiometric intensity F460/F403, Ir is the HPTS ratiometric intensity F460/F403 at each independent variable time-point t, and [ ) ] * is the averaged F460/F403 before the addition of carriers. As shown in Figure S2, the addition of metal compound 1 up to 5 μM (10 eq) did not result in any change to the HPTS ratiometric intensity F460/F403, although a slight decrease was observed with the increase of compound 1's concentration from inspection of the individual HPTS fluorescence intensities F403 (a) and F460 (b). As for the sample with the presence of NaCl (40 mM) ( Figure  In transmembrane ion transport studies using the HPTS assay, the HPTS ratiometric intensity F460/F403 is often used for reporting the overall rate of pH dissipation [5] .

Interference of vesicle lysis with HPTS response
We designed and conducted several additional experiments to determine why the fluorescence ratio of HPTS increased after vesicle lysis. The results from these control experiments indicated that this increase might be due to the different responses of HPTS when HPTS was entrapped inside vesicles and released into the external solution in this case. We had observed similar behavior of HPTS previously when potassium gluconate solutions were used as the medium. [6] (i) Comparing monensin with detergent added at the end of the experiment to collapse the pH gradient.
The transmembrane pH gradient which established by metal complex was released by two different treatments: (1) monensin (5 μM, 5 μL; 0.01 μM final concentration, 0.01 mol% carrier:lipid molar percent) via M + /H + exchange, and (2) detergent (25 µL, 11% (w%) Triton X-100 in 7:1 (v/v) H2O-DMSO) to lyse the vesicles. Note that K + is present as the counterion for the HEPES buffer. Thus, the K + /H + antiporter monensin can be used to dissipate the pH gradient. As shown in Figure S11, the transmembrane pH gradient was almost completely dissipated after the addition of monensin for all tested metal complexes 1-4. The HPTS ratiometric intensity [F460/F403] emission ratio after lysis by detergent always are higher than those by monensin. (ii) Monensin was added to dissipate any remaining pH gradient, followed by detergent added at the end of the experiment to lyse the vesicles.
In this control experiment, the pH gradient was almost completely dissipated after the addition of monensin, while further addition of detergent resulted in an increase of ratiometric intensity ( Figure S12). This finding, combined with the results in the above control experiment, indicated that the vesicle lysis affected the HPTS ratiometric response.  All above control experiment results suggested that an increase in pH after lysis is mainly due to the altered fluorescence response of HPTS after being released into the external solution. However, this interference only happened at the end of the experiment, and the ratiometric intensity after lysis was not adopted to normalize the transport data as in other reported transport experiments. Thus, this interference did not influence the results and discussion in this manuscript.

S2.1.4 Calcein leakage assays
Calcein leakage assays were conducted using POPC vesicles (mean diameter 200 nm) loaded with the fluorescence dye calcein (100 mM). The calcein-loaded POPC LUVs were prepared as follows. A chloroform solution of POPC was evaporated under vacuum and dried for at least 6 h. The thin film was hydrated by the internal solution containing calcein disodium salt (100 mM) and NaCl (100 mM) buffered to pH 7.0 with HEPES (10 mM). Then, the lipid suspension was subjected to nine freeze-thaw cycles followed by extrusion 25 times through a 200 nm polycarbonate membrane. Size exclusion chromatography using Sephadex® G-25 column and calcein-free external solution, containing NaCl (100 mM) and Na2SO4 (100 mM) buffered to pH 7.0 with HEPES (10 mM). The resulting suspension of dye-encapsulated LUVs with a mean diameter of 200 nm was diluted with the external solution to obtain a 2.5 mL lipid suspension containing a 0.1 mM lipid concentration. After the tested receptors 1-4 were added at 1 mol%, calcein fluorescence (λex = 490 nm, λem = 520 nm) was recorded at 25 °C. Detergent (25 μL) was added at t = 270 seconds to lyse the vesicle and to calibrate the assay. The fractional calcein release (FR) was normalized according to Eq. (4) as follows (with It = fluorescence intensity at time t, I0 = fluorescence intensity at time 0, and Imax = fluorescence intensity after the addition of detergent):   Figures S13 and S14 show that the addition of 1-4 (2 mol% carrier:lipid) and an increasing amount of 1 up to 10 mol% concentration has no effect on calcein release from liposomes which is signaled by an increase of calcein fluorescence emission. Interestingly, the addition of the Pt(II) complex results in a small but clearly detectable decrease of fluorescence emission, which follows a process associated with the transport of OH − from outside to inside the liposome. The increase of pH inside the liposome partially quenches the fluorescence emission of calcein leading to the observed kinetic. A smaller decrease of fluorescence emission is also observed when a 40 mM extra gradient of NaCl is applied by the addition of 25 µl of a NaCl (4 M) solution to the liposome suspension before starting the experiment (curve 1 + NaCl). However, no release of calcein in all cases, which should give an increase of fluorescence emission, is observed even at the highest concentration of ionophore tested.

S2.1.5 Carboxyfluorescein-release assays
Carboxyfluorescein-release assays were conducted using POPC vesicles (mean diameter 200 nm) loaded with the fluorescence dye 5(6)-carboxyfluorescein (CF, 50 mM). The CF-loaded POPC LUVs were prepared as follows. A chloroform solution of POPC was evaporated under vacuum and dried for at least 6 h. 5(6)-Carboxyfluorescein (CF) was initially insoluble in water and hence was dissolved in ca. 3 eq. KOH and vortexed until completely dissolved. An appropriate amount of HEPES was added to yield a ca. 50 mM CF stock in HEPES (10 mM) before final adjustment to pH 7.0 with KOH and HCl concentrated solutions. The POPC thin film was hydrated by the internal solution containing CF (50 mM) buffered to pH 7.0 with HEPES (10 mM). Then, the lipid suspension was subjected to nine freeze-thaw cycles followed by extrusion 25 times through a 200 nm polycarbonate membrane. Size exclusion chromatography using Sephadex® G-25 column and CF-free external solution containing KCl (100 mM) buffered to pH 7.0 with HEPES (10 mM). The resulting suspension of dye-encapsulated LUVs with a mean diameter of 200 nm was diluted with the external solution to obtain a 2.5 mL lipid suspension containing a 0.1 mM lipid concentration. After the tested receptors 1-4 were added, the fluorescence intensity of each sample was monitored (λex = 485 nm, λem = 520 nm) at 25 °C. Detergent (0.1% (w/v) Triton X-100, 25 μL) was added at t = 270 seconds to lyse the vesicle and to calibrate the assay. The percentage of CF fluorescence (FR) was obtained by normalizing the averaged intensities (It) against the fluorescence intensity at time 0 (I0), and fluorescence intensity after the addition of detergent (100% release, Imax) controls, according to Eq. (4) as follows: Fraction of interior volume to total volume, % in = LUV × in × A = 0.059% Fraction of exterior bulk volume to total volume, % out = -1 − ( LUV × ex × A) = 99.933% Avogadro constant, A = 6.022 × 10 23 mol −1 Literature values of the overall bilayer thickness (also known as the Luzzati thickness [7] ) and cross-sectional area of POPC (defined as area per lipid along the surface of the bilayer) are the average of reported literature values determined from both experimental and simulation methods in fully hydrated POPC lipid bilayer system, between a temperature range of 293 K to 303 K.

S2.2.2 Calculation of OH − transport
The amount of intravesicular OHtransport from the Pt(II) complex upon addition to the LUV suspension can be estimated arithmetically based on the ∆pH generated, intravesicular volume, buffer concentration, and the corrected apparent pKa' of the buffering reagent. The percentages of intravesicular volume and extravesicular bulk volume with respect to the total volume were calculated as; %Vin = 0.059% and %Vout = 99.933% for POPC LUVs with a lipid concentration of 0.1 mM and a mean diameter of 200 nm. Since the total experimental volume is 2.5 mL, the actual volumes are: Vin = 1.485 µL and Vout = 2498.320 µL. The buffering reagent HEPES has a pKa value of 7.564 at standard state (298.15 K and 0.1 MPa, at zero ionic strength). [9] The corrected apparent pK′a,T 7.450 was obtained in accordance with the experimental conditions using the following Eq. (5) and (6); [10] p !, where T is the temperature (25 °C + 273.15 =) 298.15 K, p ' ⁄ = -0.014 is the change of pKa with temperature, z = 0 is the charge of the conjugate acid, I = 0.104 is the ionic strength of the buffer solution, and = 0.5092 is a temperature-dependent constant for the Debye-Hückel relationship [11] at 25 °C. The intravesicular concentration of HPTS (1 mM) must also be considered to the overall concentration of the buffering reagent(s). Since the intravesicular apparent pKa' value of HPTS is coincidentally similar to the corrected pK'a,T 7.450 of HEPES under these experimental conditions, the subsequent calculations can be simplified by using an overall apparent pK'a,T of 7.45 for the buffering reagents, with a total concentration of 11 mM. When potassium phosphate buffer is used, an apparent pKa' value of 7.21 is adopted in calculations.
Using the pHin values, the buffer concentration of the acid and base species can be calculated based on the Henderson-Hasselbach equation [10] (1), according to Eq. (7) and (8); where Cbuffer = 11 mM is the total concentration of HPTS (1 mM) + HEPES (10 mM), with an overall pKa = 7.45 for the buffering reagents.
By substituting, (1) can be re-written as: or Based on the pHin before and after the addition of the Pt(II) complex, we can estimate the intravesicular OHtransport from the Pt(II) complex. The addition of compound 1 (1 mM, 5 μL), giving a final concentration of 2 μM (2 mol% carrier:lipid molar percent) from Figure S3, will be used as an example for the following calculations. From the above, the difference for the number of moles of HA before and after compound 1 addition is 6.72 nmol, i.e., the amount of OH − transport from compound 1.
For this instance, the number of moles of Pt(II) compound 1 (1 mM, 5 μL) added is 5 nmol, therefore the percentage of Pt(II) compound corresponding to intravesicular OH − dissociation is 134%.    ΔpHin and ∆nOH − calculated with respect to the observed HPTS ratiometric intensity F460/F403 after addition of metal compound. (b) Calculated OH − dissociation inside LUVs (%OH − in) with respect to the total amount of metal compounds.        To better illustrate the unique properties of the metal compounds, we further compared their performance with long-chain amines and long-chain acids. Fatty acids have been reported to produce acidification of pHin, caused by the fast "flip" of un-ionized of fatty acids [12] , while long-chain amines have been reported to produce alkalinization of pHin because of the fast "flip" of their un-ionized form [13] . Herein, long-chain amine 2-heptylamine (pKa = 10.70) and dodecylamine (pKa = 10.63), and fatty acid oleic acid (pKa = 5.02) were adopted as control compounds.

S2.3.2 Concentration-dependent studies
As shown in Figure S23, the addition of long-chain amines indeed induces an increase of HPTS ratiometric fluorescence intensity, indicating the increase of pHin. Dodecylamine possesses a longer chain length and displayed higher activity than heptylamine, indicating  that lipophilicity is important for their activity. It should be noted that the increase of HPTS ratiometric intensity of amines is much lower than that of compound 1 at the same concentration (2 mol% or 4 mol%, carrier:lipid molar percent). Moreover, the shape of the transport curves is very different. For long-chain amines, the diffusion of the un-ionized amine (neutral base) results in a fast jump at the beginning, but after that, the generated pH gradient generated dissipated gradually because of the slow cyclic proton transfer by amines driven by the pH gradient. In these cases, the ionized ammonium ions are totally impermeable, so there is no turnover transport. A similar phenomenon was observed in the fatty acid control, in which a fast decrease followed by a gradual increase of pHin was observed. Furthermore, the ion transport activity of long-chain amines and fatty acids under a typical transmembrane hydroxide transport test condition was also performed. As shown in Figure S24, when pHin = 7.0, pHout = 8.0, the HPTS ratiometric intensity was almost plateaued after the initial fast jump upon the addition of dodecylamine. The degree of ratiometric intensity increase was relatively higher than that observed in the pHin = 7.0, pHout = 7.0 condition because the higher pHout value results in a higher percentage of un-ionized amine. The heptylamine and oleic acid are almost inactive except for a very small pH change at the beginning. These results are very different from those observed for compound 1, in which a very high degree smooth increase of ratiometric intensity was observed at the same concentration (2 mol% or 4 mol%, carrier:lipid molar percent). All these results confirmed that the Pt(II) compounds act as hydroxide transporters with a certain turnover.

S2.4.2 Evidence for OTf dissociation
We have conducted a series of NMR experiments in different conditions to characterize the different species that might be formed. The

19
F-NMR, 31 P-NMR, 195 Pt-NMR spectrums of Pt(II) compounds were tested and collected under three conditions: I) in DMSO-d6 solution, II) in DMSO-d6/D2O (v/v, 9:1) solutions, III) with unilamellar POPC vesicles (0.1 mM) loaded with HPTS (1 mM) buffered to pH 7.0 with HEPES (10 mM) and D2O (v/v, 9:1). In the case of POPC vesicles, long accumulation times were used in order to reduce the background noise due to the low concentration of Pt(II) complexes.
The 19 F-NMR spectra were recorded on a 500 MHz spectrometer operating at 470.4 MHz, for which 0.05% trifluorotoluene was used as an internal reference resonating at −62.71 ppm. 19 F-NMR spectra of Pt(II) compound 3 (6 mM) recorded in DMSO-d6 showed the presence of only one signal at −79.33 ppm, while an upfield shifting in the peak position (at −79.37 ppm) of free triflate was observed in DMSO-d6/D2O mixture ( Figure S24). The single peak at about -79.37 ppm in the 19 F-NMR spectra is characteristic of triflate ions in the tested condition as compared to other triflate ions [14] . The 19 F-NMR spectrum of Pt(II) compound 3 (4 μM, 4 mol%, carrier:lipid molar percent) in POPC vesicles also showed a singlet at the same chemical shift value (−79.37 ppm). All these results indicated that the OTf group is fully dissociated from the Pt(II) compound in transport test conditions.  In 195 Pt-NMR spectroscopy, a triplet at -3969.69 ppm was observed for Pt(II) compound 3 (6 mM, in 0.5 mL) in DMSO-d6 solution. The 1 J( 195 Pt-31 P) value was ca. 2820 Hz, which was consistent with the 195 Pt satellites observed in the 31 P-NMR spectrum (2802 Hz). With the addition of water, this triplet signal displayed an upfield shift to -4021.57 ppm. For Pt(II) complexes, the δ(Pt) value often shifts to higher fields when coordinated to water due to the increase of the electron density in the environment of the metal atom. [15] This change, as well as the decrease in the 1 J( 195 Pt-31 P) value (ca.  All these results clearly demonstrated that the active species in POPC vesicles transport experiments are the same as the species formed in the presence of water and different from the original Pt(II) complex. Since water is the main solvent in the tested conditions and water is a small molecule that can easily approach the Pt atom on both sides of the square plane, Pt aqua complexes are considered to be the active species in this manuscript.

S2.4.3 pKa tests
We As shown in Figure S29, all these Pt(II) complexes displayed a pKa value higher than 9.0, indicating they are weakly basic. Compound 1 displayed the highest pKa value compared to other Pt(II) complexes, suggesting they can dissociate OH − easier than the other Pt(II) compounds. For Pt(II) compounds 1-3, which bear and OTf leaving groups, their OH − transport activity seems to depend on their respective pKa values. In this case, the pKa values, which determine the association rate of OH − , are considered to be representative of the rate-limiting OH − association step (step II & step IV) since OTf is a very good leaving group. On the basis of the pKa value of compound 4, it is expected to display a high transport activity. However, based on our data, compound 4, which bearing NO3 leaving groups, displayed the lowest transport activity, suggesting the solvolysis reaction step (step I) is the rate-limiting step in this case. There is no clear relevance between the transporter's pKa values and transporter activity. It should also be noted that the pKa values of Pt(II) complexes after incorporation into the phospholipid membrane might be different from those determined in solutions. Referring to the aquation reaction followed by deprotonation reactions of platinum(II) drugs like cisplatin [16] , a different form of Pt(II) species might coexist in an aqueous solution, as illustrated in Figure S30. The charge originates from the Pt(II) centers after they undergo hydrolysis with water. The kinetics of these aquation and deprotonation processes are assumed to be rapid and different species may not be isolated. Thus, the percentage of different Pt(II) species is not known in detail; however, the fast dissociation of the OTf leaving group and the pKa results of those metal compounds suggest that the predominant form of Pt(II) in an aqueous solution at a near-neutral pH is most likely to be the charged diaqua-species. The neutral diaquated Pt(II)-compound, which is the final hydrolysis product, would seem to readily traverse the membranes because of the neutral charge. We, therefore, briefly illustrate the transport mechanism by using [Pt-OH2] 2+ and Pt-OH as model species to give the reader a sense of the chemistry involved in the transport process.

S2.4.4 Measurement of membrane potential -Safranin O assays
In order to further study the OH − transport mechanism of Pt(II) complexes, the membrane potential of the liposomes during the transport process was monitored by the probe Safranin O, a membrane potential sensitive fluorescent dye that can detect the small amount of electrogenic transport possible in vesicular systems. 17 Safranin O assays were conducted using POPC vesicles (mean diameter 200 nm) which were prepared as follows. A chloroform solution of POPC was evaporated in a round-bottom flask, and the lipid film formed was dried under vacuum for at least 6 h. Then, the lipid film was hydrated by vortexing with an internal solution containing HEPES (10 mM) buffered to pH 7.0. The lipid suspension was subjected to nine freeze/thaw cycles and then extruded 25 times through a 200 nm polycarbonate membrane. The unentrapped salt was removed by size exclusion chromatography on a Sephadex® G-25 column using an external solution eluent. The lipid solution obtained after Sephadex® was diluted to a standard volume (usually 5 mL) with the external solution to obtain a lipid stock of known concentration. For each test, the lipid stock was diluted with the external buffer solution to a standard volume (2.5 mL) to afford a solution with a lipid concentration of 0.1 mM. Safranin O was added into the external buffer solution to afford a final concentration of 60 nM. The emission of Safranin O at 580 nm was monitored with an excitation wavelength of 520 nm. The emission value obtained before the addition of any transporter was used for calibration.
As shown in Figure S31, exogenous addition of the Pt(II) complexes 1-3 led to a quick decrease in the emission of Safranin O, indicating a membrane potential with a net positive charge inside the liposomes was established. Subsequently, the fluorescence intensity of Safranin O slowly increased before it returned to equilibrium with the addition of monensin (0.01 mol%). As for compound 4, a relatively slower decrease in the fluorescence intensity of Safranin O was observed. These results were consistent with the two-phase pattern (a fast jump followed by a slow change) observed in OH − transport studies. The activity trend 1 > 2 > 3 > 4 in Safranin O assay was also consistent with the activity trend in the HPTS assay.
We also tested the change of membrane potential by using Safranin O assays when long-chain amines or acids were added. It was found that there is no response in Safranin O assays upon addition of 2-heptylamine, dodecylamine, or oleic acid ( Figure S32). This result indicated that the diffusion of the neutral base or acid did not give rise to any membrane potential. However, these compounds have two Pt(II) centers that may play a role in forming a positive electric potential, and the diffusion of a species with one positively charged Pt(II) center is a possible reason for the observed results. Also, due to the membrane tension and bending rigidity, an asymmetric distribution of different Pt(II) species between the inner/outer leaflets of the LUVs could also impact the formation of a positive potential.

S2.4.5 Interpretation of the OH − transport mechanism.
Based on the above results, our proposed mechanism of increasing pHin on the addition of Pt(II) complexes is schematized in Figure  2 and Figure S33. After the spontaneous solvolysis reaction (step I), because of their low solubility in water and high hydrophobicity, the aquation product [Pt-OH2] 2+ will bind rapidly and quantitatively to the phospholipid vesicles, initially to the outer leaflet, and will deprotonate to produce a neutral complex (Pt-OH, step II). The protonation and deprotonation reactions are assumed to be fast and related to the pKa values of the Pt(II) complexes. Because of differences in relative permeability of the neutral form Pt-OH complex and the charged form [Pt-OH2] 2+ complex, the majority of nonionic Pt(II) complex must flip into the inner leaflet in response to the concentration gradient of Pt(II) complexes in the bilayer (step III). Consequently, a new acid-base equilibrium will be established in the vesicle interior, thereby releasing OH − that diffuse to the inner aqueous volume containing HPTS (step IV). This accounts for the first event in the transport process, the initial rapid increase in pH. The intravesicular alkalinization is accompanied by a rapidly established transmembrane electric potential that is stable and opposed to the chemical potential of OH − . In response to this induced electric potential, the positive charged [Pt-OH2] 2+ complexes move out of the vesicles slowly (step V) and result in a further pHin increase slowly. This second process is determined by the transport rates of charged Pt(II) aqua complexes, and the transmembrane electrical potential changed very slowly due to the relatively low permeability of the charged form Pt(II) aqua complexes. The mechanism of this transport phenomenon may be illustrated by a set of electric circuit diagrams as shown below in Figure S33. . The mechanism of OH − transport phenomena mediated by Pt(II) complexes (c) corresponding to the HEPES assay, as well as (d) corresponding transport steps. A set of (e) electric circuit diagrams to illustrate the OH − transport steps depicted in (d). 1) The intra-and extravesicular solutions are identical, and there is no charge transport/electric potential across phospholipid bilayer until Pt(II) complexes are introduced into solution; 2) the concentration gradient driven influx of neutral Pt(II) aqua complexes results in intravesicular alkalinization accompanied by a rapidly established transmembrane electric potential; 3) the potential gradient driven efflux of the charged Pt(II) aqua complexes results in a further OH − influx cycle, 4) the pH gradient and membrane potential gradient release by M + /H + exchanger or detergent.   ΔpHin and ∆nOH − calculated with respect to the observed HPTS ratiometric intensity F460/F403 after addition of metal compound. (b) Calculated OH − dissociation inside LUVs (%OH − in) with respect to the total amount of metal compounds.  ΔpHin and ∆nOH − calculated with respect to the observed HPTS ratiometric intensity F460/F403 after addition of metal compound. (b) Calculated OH − dissociation inside LUVs (%OHin − ) with respect to the total amount of metal compounds.  ΔpHin and ∆nOH − calculated with respect to the observed HPTS ratiometric intensity F460/F403 after addition of metal compound. (b) Calculated OH − dissociation inside LUVs (%OHin − ) with respect to the total amount of metal compounds.

S2.5.1 when the initial pH condition is: pHin > pHout
ΔpHin and ∆nOH − calculated with respect to the observed HPTS ratiometric intensity F460/F403 after addition of metal compound. (b) Calculated OH − dissociation inside LUVs (%OHin − ) with respect to the total amount of metal compounds. (c) The HPTS ratiometric intensity F460/F403 data after the addition of compound 1 at this concentration exceeds the maximum measurable ∆pH for this assay.  ΔpHin and ∆nOH − calculated with respect to the observed HPTS ratiometric intensity F460/F403 after addition of metal compound. (b) Calculated OH − dissociation inside LUVs (%OHin − ) with respect to the total amount of metal compounds. (c) The HPTS ratiometric intensity F460/F403 data after the addition of compound 1 at this concentration exceeds the maximum measurable ∆pH for this assay.

Metal complex addition after the external addition of anions
The vesicle stock solution was prepared as described for the standard HPTS assay, using HEPES buffer (10 mM, pH 7) without added salt for the swelling and for the size exclusion chromatography (SEC). For each test, the lipid stock was diluted with the external buffer solution to a standard volume (2.5 mL) to afford a solution with a lipid concentration of 0.1 mM. Then, an anion gradient was externally applied by the addition of 25 μL of 4 M NaX solution (X = Cl − , Br − , I − , H2PO4 − , CH3COO − , NO3 − , SO4 2− , and Glu − ). The final external concentration of NaX was around 40 mM. The cell was incubated at 298 K for 10 min. After incubation, compound 1 (2% concentration) was then added to the lipid suspension to start the experiment. The data was further normalized to get the fractional fluorescence intensity (If) by setting the average HPTS ratiometric fluorescence intensity F460/F403 before the addition of carriers to 0 and the stable emission value obtained after the addition of compound 1 (2 mol%) without the presence of anions to 100%, using the following Eq. (3): where If is the HPTS ratiometric intensity F460/F403 relative at each independent variable time-point t, [ ] * is the averaged F460/F403 before the addition of carriers. [ ] < Is the stable F460/F403 value obtained after the addition of compound 1 (2 mol%) without the presence of any anions. The relative transport rates were measured as the initial rate of fluorescence fraction changes by fitting the obtained fraction with the asymptotic function y = a -b·cx where y is the fluorescence fraction changes (%), x is time (s) and kini_r is then given by kini_r = -b·ln(c) (obtained in % s -1 ), or by fitting the obtained fraction with the two-phase exponential decay (ExpDec2) function y = A1·exp(-x/t1) + A2·exp(-x/t2) + y0, where y is the fractional fluorescence intensity (%), x is time (s) and kini_r is given by kini_r = (dy/dx)x=0 = -A1/t1-A2/t2 (obtained in % s -1 ). Then the normalized fluorescence intensity (i.e., activity) value at t = 260 s was noted from the plot. The data was further normalized to get % transport efficiency Fe by setting the fractional fluorescence intensity (If) of the DMSO sample to 0 and that of compound 1 (2 mol%) without the presence of any anions to 100.

The external addition of anions after metal complex produces a stable pH gradient
To further study the effects of anions, we conducted another anion jump experiment in which anion was externally added after a pH gradient have already been produced by compound 1. The vesicle stock solution was prepared as described for the standard HPTS assay, using HEPES buffer (10 mM, pH 7) without added salt for the swelling and for the size exclusion chromatography (SEC). For each test, the lipid stock was diluted with the external buffer solution to a standard volume (2.5 mL) to afford a solution with a lipid concentration of 0.1 mM. Compound 1 (2% concentration) was then added to the lipid suspension to start the experiment. Once stable emission was observed, 25 μL of a NaX (4 M) solution (X = Cl − , Br − , I − , H2PO4 − , CH3COO − , NO3 − , SO4 2− , and Glu − ) was added. The final external concentration of NaX was approximately 40 mM. In the concentration dependence experiment, a NaCl solution was added at different concentrations after the stable fluorescence emission of HPTS was observed. In each test, the final external concentration of the added NaCl was approximately 1, 5, 10, and 40 mM, respectively. As a control, ultra-pure (Type 1) Milli-Q® water (25 μL) was used as a control. The stable emission value obtained after the addition of compound 1 represented 100% and was used for calibration as above to obtain the fractional fluorescence intensity (If). The inhibition rates were measured as the initial rate of fluorescence fraction changes by fitting the obtained fraction after addition of anions with the asymptotic function y = a -b·cx where y is the fluorescence fraction changes (%), x is time t (s) and kini_i is then given by kini_i = −b·ln(c) (obtained in %s −1 ), or by fitting the obtained fraction with the two-phase exponential decay (ExpDec2) function y = A1·exp(−x/t1) + A2·exp(−x/t2) + y0, where y is the fractional fluorescence intensity (%), x is time t (s) and kini_r is given by kini_r = (dy/dx)x=0 = −A1/t1−A2/t2 (obtained in %s −1 ). Then the normalized fluorescence intensity (i.e., activity) value at t = 860 s was noted from the plot. The data was further normalized to get % inhibition efficiency Fie by setting the fractional fluorescence intensity (If) before adding compound 1 to 0% and setting the fractional fluorescence intensity (If) of compound 1 (2 mol%) without the presence of any anions to 100%.
where, IS(t = 860) is the normalized fluorescence intensity of a test sample with different anions at t = 860 s, I0 is the normalized fluorescence intensity before adding compound 1, and I1(t = 860) is the normalized fluorescence intensity of sample 1 without further addition of any anions at t = 860 s. Figure S43. (a) Normalized fluorescence change in HPTS emission as a function of time in the presence of compound 1 (2 mol% carrier:lipid molar percent) followed by the external addition of different concentrations of Cl − anions (as aqueous NaCl solutions); (b) Comparison of dissipation efficiency of different concentration of Cl − anions on dissipating pH gradient that compound 1 produced shown as inhibition efficiency at t = 800 s. Unilamellar POPC liposomes were loaded with HPTS (1mM) buffered to pH 7.0 with HEPES (10 mM) and dispersed in an external solution also buffered to pH 7.0 using HEPES (10 mM). Compound 1 (2 mol%) was added at t = 10 s to the lipid suspension. Once stable emission was observed, 25 µL NaCl solutions with different concentration was added at t = 360 s. The final external concentration of NaCl was approximately 1, 5, 10, and 40 mM. 25 µL water was used as a control. Each point represents the average of a minimum of 2 repeats.
To further study the membrane potential change during the anion-jump process, we also conducted an anion-'jump' experiment with the Safranin O assays. Two anions Cl − and SO4 2− , which displayed different effects on pH gradient, were studied herein. As shown in  Figure S44, and as expected, a fast decrease followed by a slight increase of fluorescence intensity with Safranin O was observed after the addition of compound 1. The external addition of anions as NaX (where X = Cl − or SO4 2− , 40 mM) resulted in a fast jump of fluorescence intensity due to the net negative charge outside the liposomes. Following the sharp initial increase, a gradual increase in fluorescence intensity was observed when Cl − was added, indicating the movement of Pt(II) species from the inner to the outer leaflet. In contrast, the addition of SO4 2− caused a very slight increase at the beginning but otherwise did not interfere with the membrane potential. Again, these results were consistent and corroborated well with the results obtained in the HPTS assays.

UV-Vis binding studies in DMSO/HEPES
To allow for a better comparison with data from the ion transport experiment, we also conducted UV-Vis binding studies in a 1:1 (v/v) DMSO/HEPES (0.01 M, pH 7) mixture. A buffer solution containing both the host (H) and the guest (G) as the NaX salt into a buffer solution of the host (Pt(II) compound 1) in a quartz cuvette thermostatted at 298 K. The UV-Vis spectra of the compound 1 in the absence and presence of an increasing concentration of the guest were measured. The absorbance values at a chosen wavelength were plotted against the guest concentration and fitted to a 1:1 binding model using a custom-written python program BindFit developed and deployed on the web www.supramolecular.org.